小鼠iPSCs诱导分化为胰岛素分泌细胞及治疗糖尿病的实验研究

发布时间：2018-02-11 15:51

本文关键词： 诱导多能干细胞胰岛素分泌细胞糖尿病　出处：《中国病理生理杂志》2016年01期 　论文类型：期刊论文

【摘要】：目的:利用3步法诱导方案,使小鼠诱导多能干细胞(induced pluripotent stem cells,iPSCs)分化为胰岛素分泌细胞(insulin-producing cells,IPCs),观察诱导效率和成熟度,并观察其对糖尿病小鼠治疗效果。方法:本实验室通过piggy Bac转座子将C57/C雄性小鼠来源胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)构建为小鼠iPSCs,利用3步法诱导方案分化为IPCs,观察细胞分化过程中的形态变化;RT-PCR和免疫荧光检测其胰岛β细胞发育相关基因和蛋白的表达;流式细胞术分析分化效率;葡萄糖刺激实验检测胰岛素和C肽分泌水平。将IPCs移植入C57/C雄性糖尿病小鼠模型左肾包膜下,监测血糖和血清中胰岛素含量28 d,观察逆转高糖血症的效果。结果:建立的3步诱导方案可以将小鼠i PSCs诱导分化为IPCs,其表达胰岛β细胞的标志性基因(Pdx1、Ngn3、Pax6和Ins2)和蛋白(Pdx1、Nkx6.1和胰岛素),在葡萄糖刺激下可分泌胰岛素和C肽。流式细胞术结果表明诱导分化的效率达28%。移植后3 d糖尿病小鼠血糖即降低至接近正常水平,血清胰岛素含量明显升高,对葡萄糖的调控能力明显增强。病理学观察IPCs细胞移植28 d后仍存活。结论:建立的3步诱导法可将i PSCs高效定向诱导分化为IPCs,明显缩短了诱导分化的时间,移植至近交系同性别小鼠体内可逆转糖尿病的高糖血症。
[Abstract]:Aim: to differentiate mouse induced pluripotent stem cells into insulin-producing cells and to observe the induction efficiency and maturity. Methods: C57 / C male mouse embryonic fibroblast derived from C57 / C mouse fibroblast was constructed into mouse iPSCs by piggy Bac transposon. Three step method was used to induce the differentiation into IPCsand the cells were observed. The morphological changes during differentiation were detected by RT-PCR and immunofluorescence. The expression of genes and proteins related to the development of islet 尾 cells was detected by RT-PCR and immunofluorescence. Flow cytometry was used to analyze the differentiation efficiency, glucose stimulation test was used to detect the secretion of insulin and C-peptide. IPCs was transplanted into the left renal capsule of C57 / C male diabetic mice. The effect of reversing hyperglycemia was observed by monitoring blood glucose and insulin content in serum for 28 days. Results: the established three-step induction protocol could differentiate mouse I PSCs into IPCs, which expressed the iconic genes of islet 尾 cell, Pdx1ngn3Pax6 and Ins2). The protein Pdx1Nkx6.1 and insulin can secrete insulin and C-peptide stimulated by glucose. Flow cytometry showed that the differentiation efficiency was 28%. 3 days after transplantation, the blood glucose of diabetic mice decreased to near normal level. The serum insulin level was significantly increased. The regulation of glucose was enhanced obviously, and the IPCs cells survived 28 days after transplantation. Conclusion: the established three-step induction method can effectively induce the differentiation of I PSCs into IPCs, and shorten the time of induction. Transplantation into inbred mice of the same sex can reverse diabetic hyperglycemia.
【作者单位】：暨南大学第二临床医学院深圳市人民医院干细胞与细胞治疗重点实验室;暨南大学医学院;
【基金】：国家自然科学基金资助项目(No.81270857) 广东省自然科学基金资助项目(No.S2013010014832) 深圳市科技计划(No.GJHZ20120618153934353)
【分类号】：R587.1;R363

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